Cancer therapy has radically changed during the last decade. Novel therapies based on the specific molecular changes that drive tumorigenesis in every patient are emerging as low toxic and more efficient alternatives to classical treatments. Thus, the targeting of the growth factor receptors, EGFR-1 and ErbB2, the estrogen nuclear receptor, or the BCR-ABL fusion protein is already part of well established clinical protocols. However, even these ideal tailored therapies fail to provide a long-term cure and can only delay the progression of the disease. Additionally to alterations in bona-fide cancer genes, in a tumor cell, multiple normal regulatory networks have been rearranged in order to adapt to the tumorigenic state. As consequence of this divergence, survival of cancer cells also depends on non-cancer genes that are essential to maintain the tumor homeostasis. This dependency generates tumor specific vulnerabilities that do not exist in normal cells and that represent opportunities for therapeutic intervention. Therefore, we postulate that interfering with these non-cancer dependencies will result in system failure, that is, the cessation of the tumorigenic state. Potential therapeutic agents attacking non-cancer nodes would have a large therapeutic window because of the non-essential nature of the targets in normal cells. Furthermore, in contrast to therapies directed against cancer genes, to escape the lethal effect tumors cells could remove/attenuate the tumorigenic primary lesion (ex. activation of an oncogene) which by itself will affect tumor fitness. Despite the relative superficial similitude among tumors, the specific alterations present in each tumor greatly influence its intrinsic characteristics. Therefore, each cancer genotype will have a distinct series of non- cancer dependencies constituting genetic synthetic lethals (GSL). In this application, we plan to identify non- cancer genes that are essential to maintain the viability of tumor cells that carry specific cancer alterations and to explore their potential use as tumor targets for tailored therapies. The systematic elimination of every gene in the genome to search for GSL represents an attractive approach to unveil tumor specific vulnerabilities. Although this process has been successfully applied in yeast to dissect cellular pathways, its use in mammals has been very limited, mainly because of the lack of proper genetic tools. However, recently RNA interference (RNAi) technology has eliminated this handicap. We have pioneered the development of RNAi-based genetic tools that greatly facilitate loss-of-function studies at a genome wide level. Thus, we plan to use our state-of-the-art RNAi-screen technology as a perturbation method to find genes that, when blocked, produce GSL effects in breast cancer cells due to the presence of bona-fide cancer genes. Here, we propose to identify the Achilles' heel of breast cancer cells using the novel concept of the non- cancer addition. Our working hypothesis completely reverses the standard strategy for finding therapeutic cancer targets which has almost exclusively focused in the inactivation of pathways activated in cancer cells. Thus, we are convinced that the successful completion of this research plan will have a considerably impact in the design of the next generation of cancer treatments. PUBLIC HEALTH RELEVANCE: One of the major challenges of modern cancer treatments is the ability to eliminate tumor cells without affecting normal ones. Cancer therapy has radically changed during the last decade. Novel therapies based on the specific molecular changes that drive tumorigenesis in every patient are emerging as low toxic and more efficient alternatives to classical treatments. However, current tailored therapies are suboptimal and, despite some exceptions, their impact in the survival of cancer patients is still modest. Here, we propose to identify this exclusive Achilles' heel of breast cancers by eliminating one by one every gene in the human genome to search for genes that, when blocked, exclusively reduce the viability of tumor cells. The successful completion of this research plan will provide us with novel targets for more efficient and less harmful breast cancer therapies and it may impact the design of future generation of cancer treatments.